DNA polymerase

Abstract

The present invention relates to a DNA polymerase possesses the properties of 1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, as compared to the case where an activated DNA is used as a substrate; 2) possessing a 3′→5′ exonuclease activity; 3) being capable of amplifying a DNA fragment of about 20 kbp, in the case where polymerase chain reaction (PCR) is carried out using λ-DNA as a template. It also relates to a DNA polymerase-constituting protein; a DNA containing the base sequence encoding thereof; and a method for producing the DNA polymerase. The present invention provides a novel DNA polymerase possessing both a high primer extensibility and a 3′→5′ exonuclease activity.

What is claimed is:  
     
       1. An isolated and purified DNA polymerase, characterized in that said DNA polymerase possesses the following properties: 
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes;  
       wherein said DNA polymerase comprises a first polypeptide and a second polypeptide, which are non-covalently bonded to form a complex,  
       wherein said first polypeptide comprises:  
       (I) the amino acid sequence encoded by a DNA of SEQ ID NO:1, or  
       (II) an amino acid sequence encoded by a DNA, the complement thereof hybridizing to the DNA of SEQ ID NO:1 under stringent conditions; and  
       wherein said second polypeptide comprises:  
       (I) the amino acid sequence encoded by a DNA of SEQ ID NO:3, or  
       (II) an amino acid sequence encoded by a DNA, the complement thereof hybridizing to the DNA of SEQ ID NO:3 under stringent conditions.  
     
     
       2. The isolated and purified DNA polymerase according to  claim 1 , characterized in that said DNA polymerase exhibits a lower error rate in DNA synthesis as compared to Taq DNA polymerase. 
     
     
       3. The isolated and purified DNA polymerase according to  claim 1 , wherein the molecular weight as determined by gel filtration method is about 220 kDa or about 385 kDa. 
     
     
       4. An isolated and purified DNA polymerase acccording to  claim 1 , characterized in that said DNA polymerase exhibits a DNA polymerase activity wherein said DNA polymerase comprises a first polypeptide and a second polypeptide, which are non-covalently bonded to form a complex, 
       wherein the molecular weight of said first polypeptide is about 90,000 Da and the molecular weight of said second polypeptide is about 140,000 Da as determined by SDS-PAGE, and  
       wherein said DNA polymerase possesses the following properties:  
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes.  
     
     
       5. An isolated and purified first polypeptide comprising the amino acid sequence as shown by SEQ ID NO:2, wherein the first polypeptide exhibits DNA polymerase activity and the following properties: 
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a second polypeptide of the amino acid sequence as shown by SEQ ID NO:4.  
     
     
       6. An isolated and purified second polypeptide comprising the amino acid sequence as shown by SEQ ID NO:4, wherein the second polypeptide exhibits DNA polymerase activity and the following properties: 
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a first polypeptide of the amino acid sequence as shown by SEQ ID NO:2.  
     
     
       7. An isolated DNA encoding a first polypeptide comprising a base sequence selected from the group consisting of: 
       (I′) a base sequence encoding the amino acid sequence as shown by SEQ ID NO:2,  
       (II′) the base sequence as shown by SEQ ID NO:1, and  
       (III′) a base sequence of a DNA, the complement thereof hybridizing to DNA of SEQ ID NO:1 under stringent conditions,  
       wherein the first polypeptide encoded by the base sequence exhibits DNA polymerase activity and the following properties:  
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl. λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a second polypeptide of the amino acid sequence as shown by SEQ ID NO:4.  
     
     
       8. An isolated DNA encoding a second polypeptide comprising a base sequence selected from the group consisting of: 
       (I′) a base sequence encoding the amino acid sequence as shown by SEQ ID NO:4,  
       (II′) the base sequence as shown by SEQ ID NO:3, and  
       (III′) a base sequence of a DNA, the complement thereof hybridizing to DNA of SEQ ID NO:3 under stringent conditions, wherein the second polypeptide encoded by the base sequence exhibits DNA polymerase activity and the following properties:  
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a first polypeptide of the amino acid sequence as shown by SEQ ID NO:2.  
     
     
       9. A method for producing a DNA polymerase, comprising the steps of: 
       culturing a transformant containing both a gene encoding a first polypeptide, which contains DNA encoding the amino acid sequence of SEQ ID NO:2, a DNA of the base sequence of SEQ ID NO:1, or a DNA, the complement thereof which hybridizes to the base sequence of SEQ ID NO:1 under stringent conditions,  
       and a gene encoding a second polypeptide, which contains DNA encoding the amino acid sequence of SEQ ID NO:4, a DNA of the base sequence of SEQ ID NO:3, or a DNA, the complement thereof which hybridizes to the base sequence of SEQ ID NO:3 under stringent conditions; and  
       collecting the DNA polymerase from the resulting culture wherein the molecular weight of said first polypeptide is about 90,000 Da and that of said second polypeptide is about 140,000 Da as determined by SDS-PAGE, and wherein said DNA polymerase possesses the following properties:  
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes.  
     
     
       10. A method for producing a DNA polymerase, comprising the steps of: 
       culturing a transformant containing a gene encoding a first polypeptide, which contains DNA encoding the amino acid sequence of SEQ ID NO:2, a DNA of the base sequence of SEQ ID NO:1, or a DNA, the complement thereof which hybridizes to the base sequence of SEQ ID NO:1 under stringent conditions,  
       and a transformant containing a gene encoding a second polypeptide, which contains DNA encoding the amino acid sequence of SEQ ID NO:4, a DNA of the base sequence of SEQ ID NO:3, or a DNA, the complement thereof which hybridizes to the base sequence of SEQ ID NO:3 under stringent conditions;  
       combining the first polypeptide contained in the resulting culture and the second polypeptide contained in the resulting culture, thereby allowing non-covalent bonding to form a DNA polymerase as a complex; and  
       collecting the DNA polymerase,  
       wherein the molecular weight of said first polypeptide is about 90,000 Da and the molecular weight of said second polypeptide is about 140,000 Da as determined by SDS-PAGE, and wherein said DNA polymerase possesses the following properties:  
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes.  
     
     
       11. A recombinant first polypeptide encoded by the isolated DNA of  claim 7 , wherein the first polypeptide exhibits DNA polymerase activity and the following properties: 
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a second polypeptide of the amino acid sequence as shown by SEQ ID NO:4.  
     
     
       12. A recombinant second polypeptide encoded by the isolated DNA of  claim 8 , wherein the second polypeptide exhibits DNA polymerase activity and the following properties: 
       1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, compared to using an activated DNA as a substrate;  
       2) possessing a 3′→5′ exonuclease activity;  
       3) amplifying a DNA fragment of about 20 kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA as a template under the following conditions:  
       PCR conditions:  
       (a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl 2 , 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase;  
       (b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes,  
       when combined with a first polypeptide of the amino acid sequence as shown by SEQ ID NO:2.